Inhibition Of The Proliferation Of Human Glioblastoma U251 Cells By Fuscocineroside A

Glioma is the most common tumor of the brain and it arises either from astrocyte or its progenitor cell.Glioblastoma multiforme(GBM)is the most frequent and aggressive kind of malignant glioma with a median survival betweent 9 and 12 months,as surgery and other current therapeutic options seem to be inadequate in curing or controlling GBM because of its nature of diffuse infiltration,relative drug resistance and insensitivity to radiation.So there is a pressing need for the identification of alternative chemopreventive or chemotherapeutic agents and strategies.Sea cucumber is widespread in Chinese seas and its main secondary metabolites of triterpene glycosides not only have the bioactivity of anti-virus,anti-fungus and anti-coagulation,but also exhibit the significant effects of anti-tumor.Fuscocineroside A is a newly isolated triterpene glycoside from the metabolites of holothuria fuscocinerea jaeger. During the screening experiments fuscocineroside A exhibited a lethal effect on tumor cells,however,its pharmacological mechanism is not clear.In this study, a laboratory research of fuscocineroside A suppressing the proliferation of human glioblastoma U251 cells is carried out and the relevant machanism is explored,which may provide theoretical evidence for the clinical application and the development of antitumor agents.1.The effects of the human glioblastoma U251 cells on growth in vitro by low dose of fuscocineroside A and the main mechanismsMethyl thiazolyl tetrazolium(MTT)proliferation assay demonstrated fuscocineroside A could markedly suppress the growth and proliferation in a dose and time-dependent manner on the human glioblastoma U251cells.The IC₅₀and IC₇₅of fuscocineroside A on U251 cells with a treatment of 48 hours were 7.8±0.3μM(**P＜0.01)and 15.4±0.5μM(**P＜0.01),respectively. Nevertheless,fuscocineroside A only exhibited slight cytotoxicity to normal nonneoplastic astrocytes.The viability of normal astrocytes only showed statistical significance at the dose of 32μM after a treatment of 72 hours(**P＜0.01).The results indicate fuscocineroside A can have a lethal effect on the human glioblastoma U251 cells under the dose during which normal astrocytes are tolerant.Compared with the negative control group,the inhibition effects on the human glioblastoma U251 cells exhibited more statistical significance when treated with a dose of over 4μM fuscocineroside A(**P＜0.01).In this study, we defined the concentration of 4μM as the treatment of low dose.We first explored the suppressing effects of low dose of fuscocineroside A on proliferation of the human glioblastoma U251 cells.Compared with the negative control group,2μM fuscocineroside A showed no suppressing effects on U251 cells(P＞0.05).After treated with 4μM fuscocineroside A for 24h,48h and 72h respectively,the survival rates of U251 cells were 80.3%,75.5%and 71.1%(*P＜0.05).Morphologic changes were observered like arrest of cell growth and vanishing of dividing phase cells under the microscope.In order to explore the possible mechanisms,we next examined the effects of fuscocineroside A on cell cycle progression with the concentration of 2μM and 4μM,respectively,and found fuscocineroside A induced a markedly increased cell percentage of G₀/G₁ phase,while the cell percentages of S phase and G₂/M phase were decreased.This indicates low dose of fuscocineroside A can induce the G₀/G₁ phase cell cycle arrest.In this process,the sub-G₁ peak,a typical marker of significant apoptosis did not appear.The results were further confirmed by the DNA agarose gel electrophoresis.Western blot showed the expression of p53,p27^kip-1and p21^waf-1proteins in the untreated human glioblastoma U251 cells were low,which were accordance with the highly aggressive character of U251 glioblastoma cells.However,these three proteins were enhanced in a time-dependent manner in fuscocineroside A-treated U251 cells,which suggested the G₀/G₁ cell cycle arrest caused by fuscocineroside A may be related with the up-regulation of p27^kip-1and p21^waf-1which is possibly mediated bp53.2.The effects of the human glioblastoma U251 cells on growth in vitro by high dose of fuscocineroside A and the main mechanismsWhen U251 cells were treated with fuscocineroside A in the concentration of 7.8μM or more higher dose,flow cytometry assay showed not only the marked G₀/G₁ phase cell cycle arrest but also the appearance of sub-G₁ peak which is the typical characteristics of apoptosis.The results indicate there are several intra-cellular signal transmitting pathways of the suppressing effects of fuscocineroside A on U251 cells.We treated the human glioblastoma U251cells with 7.8μM fuscocineroside A and found attached cells detaching from the bottom of the culture flask and turning round after 12h under the microscope. After 24h or even longer,HE staining showed cells shrank and got smaller and the cellular endochylema decreased and the nuclei aggregated and condensated. Hoechst33342 fluorescence staining showed the chromatin agglutination,nuclei condensation and nuclei fragmentation.Transmission electron microscope(TEM) showed increased lysosome,vanished microvillus,cells shrinkage,nuclei condensation and appearance of apoptotic body.TdT-mediated dUTP nick and labelling(TUNEL)technique showed that the nuclei gave out flavo-green flourescence and the apoptotic body appeared.DNA agarose gel electrophoresis also showed distinctive ladder pattern of DNA cleavage in fuscocineroside A-treated U251 cells.Western blot showed that survivin protein was strongly expressed in U251 glioblastoma cells,which was consistent in the malignancy character of U251 glioblastoma cells.However,after treated with high dose of fuscocineroside A,survivin protein content was markedly decreased.We also found high dose of fuscocineroside A can markedly enhance the expression of p53,p27^kip-1and p21^waf-1proteins,much more impressive than the results induced by low dose of fuscocineroside A.Meanwhile,caspase-3 was activated and cleaved into 19 kD active fragments which carried out the last apoptosis process.This results indicate the apoptotic death induced by fuscocineroside A in human glioblastoma U251 cells is relevant with the up-regulation of p53 and the down-regulation of survivin,both of which may be connected with the activation of caspase-3.Conclusion:Fuscocineroside A can markedly suppress the growth of human glioblastoma U251 cells,but only exhibits slight cytotoxicity to the normal nonneoplastic astrocytes.Fuscocineroside A can inhibit the proliferation and induce the G₁/S cell cycle arrest in low dose,while in the high dose fuscocineroside A can not only induce the G₁/S cell cycle arrest but also induce the apoptotic cell death.Fuscocineroside A caused G₁/S cell cycle arrest may be related with the up-regulation of p27^kip-1and p21^waf-1which is possibly mediated by p53.And the apoptotic cell death induced by fuscocineroside A in human glioblastoma U251 cells is relevant with the up-regulation of p53 and the down-regulation of survivin,and is also correlated with the activation of caspase-3.